Development of cell-based assays for cytokine receptor signaling, using an AlphaScreen SureFire assay format

The signal transducers and activators of transcription (STAT) proteins are a small family of signaling proteins that are crucial for cytokine and growth factor receptor-mediated signaling in various blood cell types. Despite their central role in immune and hematopoietic cellular regulation, there are relatively few options for monitoring receptor-mediated JAK/STAT signaling events in a cell-based format, without the need for cellular transfections or labor intensive methodology. Indeed, traditional methods such as the Western blot or ELISA remain a standard method for determining the phosphorylation status of endogenous STAT proteins. Here we present data for the rapid detection of endogenous receptor-mediated phosphorylation of multiple STAT proteins using the bead-based AlphaScreen SureFire technology. With three different cell lines (human acute monocytic leukemia THP-1 cells, human erythroleukemic TF-1 cells, and human T lymphocytic Jurkat cells), we have optimized a rapid and homogeneous methodology for monitoring endogenous, receptor-mediated signaling via STAT 1, STAT 3, or STAT 5 phosphorylation, in response to several agonists. These assays, which can be tailored for both standard research applications or high-throughput drug screening applications, afford quantitative data for receptor-mediated signaling mechanisms in an endogenous, cellular environment.